The accumulated evidence indicated that the macrophage is an essential participant in the initiation, focusing, amd amplification of antigen, mitogen, and pathogen-induced immune and inflammatory responses. Stimulated macophages secrete a peptide mediator, termed Lymphocyte Activating Factor, that may play an important, if not essential role in these cellular reactions. Lymphocyte Activating Factor has been shown to 1) enhance the proliferation and differentiation of T lymphocytes and 2) stimulate collagenase production by synovial cells from patients with rheumatoid arthritis. The major objectives of this proposal are 1) to purify Lymphocyte Activating Factor, 2) to sequence the purified peptide, and 3) to elucidate the mechanisms that regulate the production and secretion of this peptide. In the work outlined in this proposal, we will purify and sequence using established biochemical procedures, the Lymphocyte Activating Factor from a murine macrophage cell line, P388D1. The in vitro production of the mediator will be greatly enhanced by using as a stimulant, the tumor promoter and inflammatory agent, phorbol myristic acetate. In addition, we will also focus our studies on the macrophage cell line as model to elucidate the fundamental intracellular regulatory mechanisms that are involved in the production and secretion of Lymphocyte Activating Factor. Biochemical fractionation procedures will be used to determine the chemical nature of the intracellular Lymphocyte Activating Factor and inhibitors of various cellular functions will also be used to analyze the potential requirements for these functions in the production and secretion of this potent macrophage-derived peptide.